Xiaoshang Jin
Proc. Amer. Math. Soc. 152 (), 5327-5337.
Abstract, references and article information

T. A. Suslina
St. Petersburg Math. J. 35 (), 537-570.
Abstract, references and article information

The St Catherine South police are probing the fatal shooting of a fisherman in Old Harbour Bay in the parish on Sunday.

After parking his taxi cab along the sidewalk, Leon Thompson exited his vehicle and held on tightly to the tiny hands of his four small passengers. They all walked towards a makeshift bridge, and Thompson lifted each child, making four trips,...

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in “cis-mediated” style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)–linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 μm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 μm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Not the same old African story The World Today mhiggins.drupal 2 August 2022

Nollywood studio boss Mo Abudu and author Dipo Faloyin discuss how Africans are redefining how the world sees them.

Mo Abudu
EbonyLife’s latest TV drama series, Blood Sisters, was in Netflix’s global top 10. It’s a thriller and it may be slightly melodramatic because we Nigerians are melodramatic. But it deals with universal themes. Nigerians are no different to anyone else. I want EbonyLife productions increasingly to appeal to anyone in the world, even if it’s in our language. Oloture, one of our films, was about human trafficking. It was all done in pidgin English and subtitled. I watch a lot of Korean dramas and Spanish dramas that are subtitled. A good story is a good story.

Dipo Faloyin
The influence that African countries have had on the West, from music, food and film to literature, science and technology, is something people find difficult to take seriously. So, it’s good to see Netflix and other production companies take it more seriously. How has your discussion with them changed since the early days?

Mo Abudu
I have been going to an entertainment market in Cannes called Mipcom for about 12 years, and at first no one had any interest in African content. So, we focused on doing local content for local markets. Now, different communities around the world want representation in content that speaks to them. Specific countries are also saying to streamers: ‘What’s your local content strategy?’ 

Netflix was the first of the streamers to come into Africa, and it now has an Africa office. Amazon has also made inroads recently. Disney is arriving. In the United States and Britain, they just need to maintain subscriber numbers, but real growth for them is going to be in Asia and Africa. 

I’m not telling broadcasters or distributors to commission original African content as a charity project – they can make money from this. Within five days of launch, Blood Sisters registered 11 million hours of viewing on Netflix around the world. It was made on a budget five times smaller than productions outside Africa. But we need to be among the gatekeepers, too. 

Moving beyond Hollywood

Dipo Faloyin
The challenge that many creatives across Africa have is that people [elsewhere] don’t necessarily feel like they relate to this continent. They see ‘Africa’ and its cultures as very distant. Instead of intricate, specific stories, simple stories of simple people have been pushed about the continent. 

I still get asked questions like, ‘But, what should we do about Africa’s problems?’ My response is, ‘Stop seeing Africa as just a problem.’ 

Mo Abudu
I was speaking at the Qatar Economic Forum recently and the panel started off talking about the ‘problems of Africa’ – and I had to jump in and say, ‘I get you guys talking about the problems, I’m not an economist, I’m just an entrepreneur, but from an entrepreneurial perspective, we have resources – like cobalt from the Democratic Republic of Congo that’s in all of our mobile phones.’ 

The problem is, we ship out all our resources and by the time they come back to us, they are 10-times more expensive than we can afford. I keep saying that they need to know they need us as much as we need them.
 
Dipo Faloyin
There are certainly issues within the continent like there are everywhere else; but more accurate stories will help people have a better sense of the context in which so many communities and their lives have been built up. 

Mo Abudu
The West doesn’t seem to have any interest in making films about Africa unless it’s about the worst of Africa: the slave trade, the Rwandan genocide, blood diamonds. That seems to be what has defined us. 

Dipo Faloyin
If you ask most people around the world to close their eyes and picture Africa, two images will come up: safari, and poverty and strife. Until the age of 12, I grew up in Lagos, a metropolis with no wild animals running around. There are slums, of course, but also traffic, shopping centres and overpriced restaurants. 

Writers who pitch ideas to Vice.com where I work often still don’t differentiate African countries. They’ll say, ‘There’s been a coup in Mali. Why can’t Africa get its head around democracy?’, and I remind them a small minority of countries on the continent is under any form of authoritarian rule. 

It’s frustrating that this perception hasn’t changed. For us to break through we need big cultural institutions – Hollywood, museums, literature – to allow people from across the regions to tell these stories. We are rarely portrayed as protagonists and forward thinkers. But I’m excited for the future.

Sudan’s gold boom: Connections to conflict and transnational impacts 7 December 2022 — 2:00PM TO 3:30PM Anonymous (not verified) 24 November 2022 Online

At this event, experts will discuss Sudan’s gold sector, its connections to conflict, and transnational impacts. 

At this webinar panellists will discuss Sudan’s gold sector, its connections to conflict, and transnational impacts.

Sudan is one of the largest gold producers on the continent, with the industry constituting Sudan’s foremost source of hard currency since the secession of South Sudan in 2011 and resulting loss of oilfields.

The gold rush that has ensued has had important implications for domestic and transnational conflict dynamics. Military actors and armed groups have sought control of gold-producing areas in the peripheries and to capitalize on the flow of labour migrants, against a wider backdrop of conflict partly stemming from contestation for control between central and local actors.

International interests are prominent, including increased Russian involvement in the sector, while gold smuggling has also interlaced with mercenary activity in neighbouring CAR, Chad and Libya.
 
At this event, panellists will discuss Sudan’s gold trade, its connections to conflict, and transnational impacts, including the international politics of Sudan’s gold extraction and role of armed groups. It will also explore the environmental and socio-economic dimensions of gold in Sudan’s border areas. 
 
This roundtable is an output of the Cross-Border Conflict: Evidence, Policy and Trends (XCEPT) research programme, funded by UK Aid from the UK government.
 

Fighting over ‘white gold’: Sesame in Ethiopia and Sudan Expert comment LJefferson 3 April 2023

The supply chain of a seemingly innocuous cash crop – sesame – has intersected with transnational conflict dynamics, exacerbating tensions between Ethiopia and Sudan.

Late 2020 saw the beginning of the devastating war in Tigray and the occupation of a disputed region on the Ethiopia–Sudan border – Al Fashaga – by the Sudanese army. These shocks disrupted settled patterns of land ownership and control in both Ethiopia’s volatile north and Sudan’s borderlands, historically the heart of the sesame and oilseed production that is economically vital to both countries.

These seemingly harmless cash crops are now embedded in local, subnational and national political contestations in both countries. Sesame value chains are being reshaped, with power and profits being used to entrench the grip of political and armed actors who are reinforcing new patterns of land control and driving informal and illicit trade – impacting the coping mechanisms of local communities and threatening to fuel further conflict.

Regional rivalries drive contestation over the Ethiopia supply chain

Internal borders between most of Ethiopia’s regions are marked by boundary disputes, which often degenerate into violent conflict. The most important is between the Tigray and Amhara regions. Since the war began in 2020, the Amhara region has annexed vast areas of western and southern Tigray, which the Amhara region claims were taken from them by Tigray 30 years ago, after the Tigray People’s Liberation Front (TPLF) dominated ethnic coalition came to power.

Ethiopia’s exports of spices, oilseeds and pulses brought in over half a billion dollars in 2021, roughly a quarter of the country’s total export revenues and second only to coffee. The sector has been rocked by the war in the north, which accounted for much of Ethiopia’s sesame production, with an estimated 500,000 hectares of sesame fields taken out of cultivation during the 2021 growing season. Conflict has exacerbated a steady decline in formal revenues from sesame exports, dropping over $115 million from 2016 to 2021.

Alongside falling production, the previously integrated value chain has been disrupted and decentralized by political fragmentation and land competition between Amharas and Tigrayans. Before the war, the agricultural sector in Western Tigray/Welkait was dominated by Tigrayan business interests, through the TPLF’s regional endowment fund EFFORT, a business conglomerate including subsidiaries such as Guna Trading House, and Hiwot Agricultural Mechanization.